Project Summary The objective of this two-year R21 project is to explore a very new approach to probing the dynamics of enzyme activity based on recently-discovered electronic properties of proteins. The long-term goal is to create a low-cost, high speed single-molecule genome sequencer with long reads, requiring no dyes or labels, with direct electronic readout. The technology has the potential for rapid reads (on the order of an hour per genome) using an integrated circuit chip. If this potential were to be fully realized, then use of genome sequencing in the clinic with near real-time feedback could become a reality. Initial data shows that high signal to noise measurement of protein fluctuations appears to be possible, and suggests that at least some of the fluctuations are associated with the function of the protein. However, these studies are limited by a great deal of variability owing to uncertainty about the chemical nature of contacts to the proteins. Therefore, our first aim is to engineer polymerases with built-in contacts, designed to self-assemble into a measurement device. We will test the construct with a scanning tunneling microscope, and, in the process, obtain design parameters for a sequencing device. Our second aim is to test at least one structure in a solid-state device, to check the validity of data obtained in a scanning tunneling microscopy. If successful, the data collected would lay the groundwork for a larger effort to scale up and improve accuracy to achieve a viable sequencing platform with the properties described above.